Research is proposed to 1) determine what mechanisms of peptide influence on the EMP of 4Fe-4S clusters functions in iron-sulfur proteins, and 2) determine if cysteinyl sulfur atoms exposed to the solvent in C. pasteurianum ferredoxin provide the pathway for electron transfer. NMR and resonance Raman experiments are described to determine if the reduction potential is altered by peptide constraint of the iron-sulfur cluster to specific geometries. These studies involve primarily A. vinelandii Fd I. To determine the extent to which NH...S hydrogen bonds to the 4Fe-4S cluster influence the reduction potential, NMR and EPR studies, in concert with reduction potential measurements of deuterated samples of C. Chromatium HiPIP, and A. vinelandii Fd I are proposed. As a partial test of electrostatic effects of the peptide, NMR spectra of R. gelatinosa and Chromatium HiPIPs will be recorded. Finally, the origin of the shift in Emp in C. acidi-urici Fd observed upon replacement of the single amino acid Tyr 2 with either His or Trp will be determined using NMR and EPR. The possibility of crysteinyl sulfur atoms providing the pathway for electron transfer in C. pasteurianum ferredoxin will be tested by use of Cr(II) to label the site of electron transfer, and by treatment of the protein with (terpy)PtCl ion and Ru(Nh3)5H2O2 ion to modify the exposed cysteinyl sulfur atoms. By a series of biological assays and kinetic studies it will be possible to determine if cysteinyl sulfur atoms provide the pathway for electron transfer in this protein.